Water immersion objective lens

To assess the biofilm spatial organization on CS-PLA surfaces, single- and dual-species biofilms of S. aureus and P. aeruginosa were observed by CSLM [78 (link),79 (link)]. First, 24 h biofilms of S. aureus and S. aureus + P. aeruginosa formed on PLA and functionalized surfaces were counterstained with 6 µM SYTO^®9 (Thermo Fisher Scientific, USA). S. aureus, P. aeruginosa and S. aureus + P. aeruginosa biofilms were then observed using a 40× water immersion objective lens (Leica Microsystems, Germany) in an inverted microscope Leica DMI6000-CS with 488 nm argon and 633 nm helium-neon lasers. The emitted fluorescence was recorded within the ranges of 500 to 580 nm and 640 to 730 nm to collect the SYTO^®9 and mCherry emission fluorescence, respectively. A minimum of five stacks of horizontal plane images (512 × 512 pixels, corresponding to 387.5 µm × 387.5 µm) with a z-step of 1 µm was acquired for each biofilm sample.
Three-dimensional (3D) projections of biofilm structures were reconstructed from the CLSM acquisitions using the blend mode of the “Easy 3D” function of IMARIS 9.1 software (Bitplane, Zurich, Switzerland). The plug-in COMSTAT2 associated with the ImageJ software was used to determine the biovolume (µm³·µm⁻²) and biofilm thickness (µm) [80 (link)].

The spatial structure of single- and dual-species biofilms of P. aeruginosa and S. aureus on PDMS and GNP/PDMS surfaces was assessed by CSLM as described by Lima et al. [7 (link)]. S. aureus and P. aeruginosa + S. aureus biofilms were first counterstained with 6 µM SYTO^®9 (Invitrogen Life Technologies, Alfagene, Portugal). All biofilm samples were then observed using a 40× water immersion objective lens (Leica Microsystems, Wetzlar, Germany) in an inverted microscope Leica DMI6000-CS with 488 nm argon and 633 nm helium-neon lasers. Image stacking was acquired with a 1 μm thickness for each sample at a minimum of five random fields. Image processing was performed using the IMARIS 9.1 software package (Bitplane, Zurich, Switzerland) for modelling in two and three dimensions [55 (link)]. The CLSM acquisitions were analyzed by the plug-in COMSTAT2 associated with the ImageJ software to determine biofilm structural parameters such as biovolume (µm³ µm⁻²) and average thickness (µm) [56 (link)].